CN102552309A - Application and preparation method of gold hyaluronic acid - Google Patents

Application and preparation method of gold hyaluronic acid Download PDF

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CN102552309A
CN102552309A CN2010106063561A CN201010606356A CN102552309A CN 102552309 A CN102552309 A CN 102552309A CN 2010106063561 A CN2010106063561 A CN 2010106063561A CN 201010606356 A CN201010606356 A CN 201010606356A CN 102552309 A CN102552309 A CN 102552309A
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hyaluronic acid
gold
acid gold
solution
halogenation
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CN102552309B (en
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朱湛
郭炳南
窦晶晶
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Beijing Institute of Technology BIT
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Beijing Institute of Technology BIT
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Abstract

The invention relates to an application and a preparation method of gold hyaluronic acid, belonging to the field of synthesis of a novel medicament for treating tumors. Specific to the defects that gold hyaluronic acid is not applied to the preparation of a tumor treating medicament and that a gold halide and sodium hyaluronic acid or hyaluronic acid are used for preparing gold hyaluronic acid by reacting in the prior art, the invention provides an application of gold hyaluronic acid to the preparation of a tumor treating medicament, particularly provides an application of the gold hyaluronic acid to the preparation of a cancer treating medicament, and more particularly provides an application of the gold hyaluronic acid to the preparation of a liver cancer treating medicament which has suppression effects on various cancer cells and particularly has a remarkable suppression effect on liver cancer cells. The invention further provides a method for preparing gold hyaluronic acid by reacting gold halide with sodium hyaluronic acid or hyaluronic acid. The preparation method has the advantages of easiness, high yield and low cost.

Description

Purposes of a kind of hyaluronic acid gold and preparation method thereof
Technical field
The present invention relates to purposes of a kind of hyaluronic acid gold and preparation method thereof; Specifically; Relate to the application of a kind of hyaluronic acid gold as preparation medicine for treating tumor thing, the particularly application of conduct preparation treatment cancer drug, more particularly treat the application of liver-cancer medicine as preparation; Also relate to a kind of method, belong to the synthetic field of the new drug of treating tumor with halogenation gold and hyaluronate sodium or hyaluronic acid prepared in reaction hyaluronic acid gold.
Background technology
Hyaluronic acid (hyaluronic acid; Be called for short HA); Having another name called glass acid, is the multiple glycosaminoglycans of disaccharidase unit that is connected with β-1,4 glycosidic bonds through β-1,3 by glucuronic acid and N-acetyl group glucamine, and said hyaluronic structure is suc as formula shown in (I).
Hyaluronic acid is present between the multiple histiocyte of spinal animals in the matter; Have the viscoelasticity of height, unique moisture retention, good biocompatibility and degradability; And have physiological actions such as nutrition, reparation and prevention damage; The hyaluronic acid oligose fragment all plays an important role with regulating in the intracellular activity in the structure of keeping extracellular matrix, is widely used in fields such as surgery, ophthalmology, department of dermatologry and beauty treatment, cosmetics.Because in most of malignant tumor of research at present, all find the little normal structure that is higher than of hyaluronic expression, and the overexpression that detects hyaluronic specific receptor CD44, so hyaluronic acid is also as some cancer therapy drug targeting mediations.After hyaluronic acid and paclitaxel were carried out esterification, the active effect of vitro inhibition growth of bladder cancer cells obviously was better than paclitaxel.Oral experiment proof hyaluronic acid oligose fragment belongs to innocuous substance, and after rat acute toxicity showed oral 500mg/kg hyaluronic acid oligose fragment, no antigen did not have irritated reaction, no teratogenesis, mutagenesis and carcinogenesis.
Gold is available medical material.As far back as B.C. 2500, China just had with the record of gold as multiple medicine and nutriment.The pure and mild medical officials of imperial physician Liu of the Ming Dynasty went through 65 years through reagent, were divided into six big types to 5611 flavor medicines, wherein acute poison 132 flavors, and slow poison 911 flavors are not gone into medical material 565 flavors, and weak medical material is 3559 flavors, as medical material 301 flavors, available medical material 143 flavors.Gold is one of 143 herbs flavor available (see Liu Hong Zhang, Liu gushing forward, three-drug.China friendship publishing house, in February, 2007 Beijing first impression.397～418 pages.)。Nearly tens annuities also begin to be used for clinical.The active anticancer that has now found that tetraphenylporphyrin gold (III) is stronger 100 times than clinical anticarcinogen cisplatin commonly used.On structure, the Pt (II) of Au (III), cisplatin has (d 8) wait electronic structure.
United States Patent (USP) (United States Patant; 4784991; 1988; 11～15.) once mentioning the hyaluronic acid gold has antibacterial activity, but does not see the report of hyaluronic acid gold as the application of the medicine of preparation treatment tumor, does not also see with the report of halogenation gold with the method for hyaluronate sodium or hyaluronic acid prepared in reaction hyaluronic acid gold.
Summary of the invention
Do not prepare the application of medicine for treating tumor thing and still do not have use halogenation gold and hyaluronate sodium or the golden defective of hyaluronic acid prepared in reaction hyaluronic acid to still there being the conduct of hyaluronic acid gold in the existing skill; An object of the present invention is to provide of the application of a kind of hyaluronic acid gold as preparation medicine for treating tumor thing; Particularly treat the application of cancer drug as preparation; More particularly, the liver cancer cell multiplication had very high suppression ratio as the application of preparation treatment liver-cancer medicine.
Another object of the present invention provides a kind of method for preparing of hyaluronic acid gold, and said method for preparing is meant with the method for halogenation gold with hyaluronate sodium or hyaluronic acid prepared in reaction hyaluronic acid gold.
Technical scheme of the present invention is following:
A kind of hyaluronic acid gold particularly as the application of preparation treatment cancer drug, more particularly as the application of preparation treatment liver-cancer medicine, has very high suppression ratio to the liver cancer cell multiplication as the application of preparation medicine for treating tumor thing.The chemical formula of said hyaluronic acid gold is (C 14H 20O 11N) 3Au5H 2O, each hyaluronic acid gold molecule is represented with the disaccharidase form.
A kind of method for preparing of hyaluronic acid gold, concrete method for preparing step is following:
(1) Sodium Hyaluronate or hyaluronic acid are placed deionized water; Stirring at room solution makes it to dissolve fully and is transparence; Obtain Sodium Hyaluronate or hyaluronic acid solution; After the halogenation gold is dissolved in deionized water, obtain the halogenation gold solution, said halogenation gold solution is joined in said Sodium Hyaluronate or the hyaluronic acid solution; Stir; Room temperature reaction 8～24h stops to stir, and obtains reaction solution; Or the halogenation gold is dissolved in the deionized water, under stirring at room, add Sodium Hyaluronate or hyaluronic acid, till room temperature continues to be stirred to dissolving fully, stop to stir then, obtain reaction solution;
(2) said reaction solution is poured in the dehydrated alcohol below 0 ℃, produced thread deposition;
(3) said thread deposition is washed 3～4 times with decantation with the dehydrated alcohol below 0 ℃ or with using absolute ethanol washing behind the said thread sedimentation and filtration, the thread deposition after obtaining washing;
(4) the thread sedimentation and filtration after said the washing is gone out and below 40 ℃, after drying under the 0.09MPa vacuum condition, promptly obtain hyaluronic acid gold of the present invention.
Wherein, the halogenation gold is 1: 1 with the ratio of hyaluronate sodium or hyaluronic amount of substance in the step (1), and said halogenation gold refers to AuX 3, X be Cl, Br, I or F one of them;
The halogenation gold is following with the reaction equation of hyaluronate sodium:
3NaHA+3AuX 3→Au(HA) 3+3NaX+2AuX 3；
The dehydrated alcohol purity of using in the said method for preparing as AG or more than.
Beneficial effect
1. hyaluronic acid gold of the present invention is through external tetramethyl azo azoles salt trace enzyme reaction colorimetric method for determining, and is inhibited to multiple cancerous cell;
2. hyaluronic acid gold of the present invention has the obvious suppression effect to suppressing the liver cancerous cell, when the dosage of said hyaluronic acid gold is 600 μ g/mL, to people's liver cancerous cell BEL7402 inhibition of proliferation rate up to 96.5%;
3. the method for preparing of hyaluronic acid gold of the present invention is simple, and output is high, and cost is low.
Description of drawings
The infrared spectrum of the hyaluronic acid gold that Fig. 1 makes for the embodiment of the invention 1.
The proton nmr spectra of the hyaluronic acid gold that Fig. 2 makes for the embodiment of the invention 1 ( 1The HNMR spectrogram).
Energy dispersion type x-ray fluorescence spectrometry (EDX) figure of the hyaluronic acid gold that Fig. 3 makes for the embodiment of the invention 1.
The specific embodiment
In order to prove absolutely the mode of characteristic of the present invention and embodiment of the present invention, provide embodiment below.
Employed dehydrated alcohol is AG among embodiment 1 and the embodiment 2, and reaction equation is following:
3NaHA+3AuCl 3→Au(HA) 3+3NaCl+2AuCl 3
Embodiment 1
(1) get 0.250g (0.143mmol) hyaluronate sodium powder and place flask, add deionized water 40mL, stirring at room solution makes it to dissolve fully and is transparence, obtains sodium hyaluronate solution, with tetrachloro alloy acid (AuCl 3) 0.059g (0.143mmol) is dissolved in the 50mL deionized water, obtains the tetrachloro alloy acid solution, and said tetrachloro alloy acid solution is joined in the said sodium hyaluronate solution, stirs room temperature reaction 12h; Stop to stir, obtain reaction solution;
(2) said reaction solution is poured in 0 ℃ the dehydrated alcohol of 2 times of volumes of reaction solution, produced thread deposition;
(3) said thread deposition is washed 4 times the thread deposition after obtaining washing with decantation with 0 ℃ the dehydrated alcohol of 150mL;
(4) the thread sedimentation and filtration after said the washing is gone out and at 40 ℃, behind the dry 24h, promptly get 0.225g hyaluronic acid gold according to the invention under the 0.09MPa vacuum condition, in the reactant hyaluronate sodium, the yield of product hyaluronic acid gold is 76%.
The product that embodiment 1 is prepared carries out structural characterization, and the result is following:
(1) The results of FT-IR: can know according to Fig. 1, at 3438cm -1The place is O-H stretching vibration, 2922cm -1The place is C-H symmetrical stretching vibration, 1734cm -1The place is the asymmetrical stretching vibration of the two keys of C=O, 1610cm -1Be OCO asymmetrical stretching vibration, 1419cm -1Be OCO symmetrical stretching vibration, 1647cm -1The place is amide I band (CO links to each other), 1635cm -1The place is C=O stretching vibration on the amide, 1554cm -1The place is amide II band (NH links to each other), 1318cm -1The place is amide III band, 1037cm -1The place is the C-O-C stretching vibration; Compare with hyaluronate sodium, 1734cm occurred -1The asymmetrical stretching vibration of the two keys of the C=O of place;
(2) elementary analysis value (C 14H 20O 11N) 3Au5H 2O (measured value/% (theoretical value/%)): C:35.43 (35.48), H:4.97 (4.96), N:2.95 (2.96);
(3) proton nmr spectra result 1HNMR (D 2O) result: δ H:3.314～3.926 (H in the hyaluronic acid on the hexatomic ring), 1.946 (S 3H CH 3), 4.511 (m-CH 2OH 2H);
(4) energy dispersion type x-ray fluorescence spectrometry (EDX) analysis result: contain the gold dollar element in the characteristic peak demonstration product in the EDX collection of illustrative plates, further confirm to successfully synthesize the hyaluronic acid gold.
Can know that through the said structure characterization result product that embodiment 1 prepares is the hyaluronic acid gold, chemical formula is (C 14H 20O 11N) 3Au5H 2O, each hyaluronic acid gold molecule is represented with the disaccharidase form.
Embodiment 2
(1) tetrachloro alloy acid 0.059g (0.143mmol) is dissolved in the 50mL deionized water; Under stirring at room, slowly add 0.25g (0.143mmol) hyaluronate sodium powder, then after room temperature continues to be stirred to dissolving fully; Continue to stir 8h and react completely, obtain the transparence reaction solution;
(2) under strong agitation, in said reaction solution, add 0 ℃ dehydrated alcohol of 3 times of volumes, produce thread deposition;
(3) with behind the said thread sedimentation and filtration, with 0 ℃ the absolute ethanol washing of 150mL 4 times, the thread deposition after obtaining washing;
(4) the thread sedimentation and filtration after said the washing is gone out and at 40 ℃, behind the dry 24h, promptly obtain 0.248g hyaluronic acid gold of the present invention under the 0.09MPa vacuum condition, in the reactant hyaluronate sodium, the yield of product hyaluronic acid gold is 84%.
The product that embodiment 2 is prepared carries out structural characterization, and the result is following:
(1) The results of FT-IR: 3439cm -1The place is O-H stretching vibration, 2921cm -1The place is C-H symmetrical stretching vibration, 1733cm -1The place is the asymmetrical stretching vibration of the two keys of C=O, 1611cm -1Be OCO asymmetrical stretching vibration, 1418cm -1Be OCO symmetrical stretching vibration, 1646cm -1The place is amide I band (CO links to each other), 1634cm -1The place is C=O stretching vibration on the amide, 1553cm -1The place is amide II band (NH links to each other), 1319cm -1The place is amide III band, 1036cm -1The place is the C-O-C stretching vibration.Compare with hyaluronate sodium, 1733cm occurred -1The asymmetrical stretching vibration of the two keys of the C=O of place;
(2) elementary analysis value (C 14H 20O 11N) 3Au5H 2O (measured value/% (theoretical value/%)): C:34.13 (35.48), H:5.02 (4.96), N:3.11 (2.96);
(3) proton nmr spectra 1HNMR (D 2O) result: δ H:3.314～3.926 (H in the hyaluronic acid on the hexatomic ring), 1.496 (S 3H CH 3), 4.511 (m-CH 2OH 2H);
(4) energy dispersion type x-ray fluorescence spectrometry (EDX) analysis result: contain the gold dollar element in the characteristic peak demonstration product in the EDX collection of illustrative plates, further confirm to successfully synthesize the hyaluronic acid gold.
Can know that through the said structure characterization result product that embodiment 2 prepares is the hyaluronic acid gold, chemical formula is (C 14H 20O 11N) 3Au5H 2O, each hyaluronic acid gold molecule is represented with the disaccharidase form.
The principle of embodiment 3～8 is: the dehydrogenase in the living cells can be reduced into water-fast bluish violet product (formazan) with tetrazolium salts, and is deposited in the cell, and dead cell does not have this function.Dimethyl sulfoxide (DMSO) can will be deposited in the bluish violet product stripping in the cell, and the solution colour depth is directly proportional with contained formazan amount, measures its absorbance (OD) with ELIASA, and absorbance is directly proportional with shade.
Employed 1640 cell culture mediums are produced by U.S. Gibico company; Cell culture is used NBCS, is produced by Hangzhou biological engineering material company limited; Thiazolyl blue (MTT) is produced by Solarbio company; Dimethyl sulfoxide is produced by Beijing chemical reagents corporation; The enzyme mark detects to be produced by U.S. Costar company with 96 orifice plates; The ELIASA model: 450, U.S. BIO-RAD company produces; Calculate IC 50Software be Sigmaplot 8.0.
The original seed of embodiment 3 and 4 employed BEL7402 human liver cancer cells, embodiment 5 and 6 employed A549 human lung carcinoma cells and embodiment 7 and 8 employed human cervical carcinoma Hela cells is from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's (basic institute coordinates) cell bank; BEL7402 human liver cancer cell that uses and A549 human lung carcinoma cell are the institute of Materia Medica,Chinese Academy of Medical Sciences cultivation of going down to posterity, and human cervical carcinoma Hela cell is the cultivation of going down to posterity of Beijing Institute of Technology's school of life and health sciences.
Embodiment 3
(1) the hyaluronic acid gold that embodiment 1 is prepared adds a little dimethyl sulfoxide earlier, adds phosphate buffer again it is dissolved fully, obtains the mother solution of hyaluronic acid gold.With 1640 culture medium that contain 10% serum, the mother solution of said hyaluronic acid gold is diluted to the working solution concentration of hyaluronic acid gold;
(2) the BEL7402 human liver cancer cell is processed cell suspending liquid, add 96 well culture plates (100 μ L/ hole), place 37 ℃, 5%CO by finite concentration 2Cultivate 24h in the incubator under the condition.Add the working solution of said hyaluronic acid gold, its final concentration is respectively: 0 μ g/mL, 5 μ g/mL, 50 μ g/mL, 200 μ g/mL and 600 μ g/mL (4 multiple holes of each concentration).Discard culture fluid behind the effect 72h, every hole adds the RPMI-1640 (containing 10% serum) that 100 μ L contain 0.5mg/mL MTT, places 37 ℃, 5%CO 2Produce blue crystallization after cultivating 4h in the incubator under the condition, abandon liquid, every hole adds dimethyl sulfoxide 150 μ L; Jolt 10min under the room temperature; Blue crystallization is dissolved fully, and to detect wavelength 570nm, reference wavelength 655nm measures every hole OD value in Bio-Rad 450 type ELIASAs.
Do not add cell and do not add hyaluronic acid gold and be background, be 100% and suppress; Only add cell and do not add the negative contrast of hyaluronic acid gold, be 0% and suppress.
Hyaluronic acid gold suppression ratio %=(negative control OD-hyaluronic acid gold OD)/(negative control OD-background OD) * 100%.
As shown in table 1, suppression ratio wherein and standard deviation are percentile average of the inhibition of each parallel hole in every group of dosage and standard deviation.Result's matter acid gold that shows transparency has the propagation of BEL7402 human liver cancer cell and suppresses active, and especially the propagation to the BEL7402 human liver cancer cell has obvious inhibiting activity under high dose.
Table 1 hyaluronic acid gold is to the influence of BEL7402 human liver cancer cell propagation
Figure BSA00000399255800061
Embodiment 4
(1) the hyaluronic acid gold that embodiment 2 is prepared adds a little dimethyl sulfoxide earlier, adds phosphate buffer again it is dissolved fully, obtains the mother solution of hyaluronic acid gold.With 1640 culture medium that contain 10% serum, the mother solution of said hyaluronic acid gold is diluted to the working solution concentration of hyaluronic acid gold;
(2) the BEL7402 human liver cancer cell is processed cell suspending liquid, add 96 well culture plates (100 μ L/ hole), place 37 ℃, 5%CO by finite concentration 2Cultivate 24h in the incubator under the condition.Add the working solution of hyaluronic acid gold, its final concentration is respectively: 0 μ g/mL, 5 μ g/mL, 50 μ g/mL, 200 μ g/mL and 600 μ g/mL (4 multiple holes of each concentration).Discard culture fluid behind the effect 72h, every hole adds the RPMI-1640 (containing 10% serum) that 100 μ L contain 0.5mg/mL MTT, places 37 ℃, 5%CO 2Produce blue crystallization after cultivating 4h in the incubator under the condition, abandon liquid, every hole adds dimethyl sulfoxide 150 μ L; Jolt 10min under the room temperature; Blue crystallization is dissolved fully, and to detect wavelength 570nm, reference wavelength 655nm measures every hole OD value in Bio-Rad 450 type ELIASAs.
Do not add cell and do not add hyaluronic acid gold and be background, be 100% and suppress; Only add cell and do not add the negative contrast of hyaluronic acid gold, be 0% and suppress.
Hyaluronic acid gold suppression ratio %=(negative control OD-hyaluronic acid gold OD)/(negative control OD-background OD) * 100%.
As shown in table 2, suppression ratio wherein and standard deviation are percentile average of the inhibition of each parallel hole in every group of dosage and standard deviation.Result's matter acid gold that shows transparency has the propagation of BEL7402 human liver cancer cell and suppresses active, and especially the propagation to the BEL7402 human liver cancer cell has obvious inhibiting activity under high dose.
Table 2 hyaluronic acid gold is to the influence of BEL7402 human liver cancer cell propagation
Embodiment 5
(1) the hyaluronic acid gold that embodiment 1 is prepared adds a little dimethyl sulfoxide earlier, adds phosphate buffer again it is dissolved fully, obtains the mother solution of hyaluronic acid gold.With 1640 culture medium that contain 10% serum, the mother solution of said hyaluronic acid gold is diluted to the working solution concentration of hyaluronic acid gold;
(2) the A549 human lung carcinoma cell is processed cell suspending liquid, add 96 well culture plates (100 μ L/ hole), place 37 ℃, 5%CO by finite concentration 2Cultivate 24h in the incubator under the condition.Add the working solution of hyaluronic acid gold, its final concentration is respectively: 0 μ g/mL, 5 μ g/mL, 50 μ g/mL, 200 μ g/mL and 600 μ g/mL (4 multiple holes of each concentration).Discard culture fluid behind the effect 72h, every hole adds the RPMI-1640 (containing 10% serum) that 100 μ L contain 0.5mg/mL MTT, places 37 ℃, 5%CO 2Produce blue crystallization after cultivating 4h in the incubator under the condition, abandon liquid, every hole adds dimethyl sulfoxide 150 μ L; Jolt 10min under the room temperature; Blue crystallization is dissolved fully, and to detect wavelength 570nm, reference wavelength 655nm measures every hole OD value in Bio-Rad 450 type ELIASAs.
Do not add cell and do not add hyaluronic acid gold and be background, be 100% and suppress; Only add cell and do not add the negative contrast of hyaluronic acid gold, be 0% and suppress.
Hyaluronic acid gold suppression ratio %=(negative control OD-hyaluronic acid gold OD)/(negative control OD-background OD) * 100%.
As shown in table 3, suppression ratio wherein and standard deviation are percentile average of the inhibition of each parallel hole in every group of dosage and standard deviation.Result's matter acid gold that shows transparency has the propagation of A549 human lung carcinoma cell and suppresses active, and especially the propagation to the A549 human lung carcinoma cell has the edge to suppress active under high dose.
Table 3 hyaluronic acid gold is to the influence of A549 human lung carcinoma cell propagation
Figure BSA00000399255800081
Embodiment 6
(1) the hyaluronic acid gold that embodiment 2 is prepared adds a little dimethyl sulfoxide earlier, adds phosphate buffer again it is dissolved fully, obtains the mother solution of hyaluronic acid gold.With 1640 culture medium that contain 10% serum, the mother solution of said hyaluronic acid gold is diluted to the working solution concentration of hyaluronic acid gold;
(2) the A549 human lung carcinoma cell is processed cell suspending liquid, add 96 well culture plates (100 μ L/ hole), place 37 ℃, 5%CO by finite concentration 2Cultivate 24h in the incubator under the condition.Add the working solution of hyaluronic acid gold, its final concentration is respectively: 0 μ g/mL, 5 μ g/mL, 50 μ g/mL, 200 μ g/mL and 600 μ g/mL (4 multiple holes of each concentration).Discard culture fluid behind the effect 72h, every hole adds the RPMI-1640 (containing 10% serum) that 100 μ L contain 0.5mg/mL MTT, places 37 ℃, 5%CO 2Produce blue crystallization after cultivating 4h in the incubator under the condition, abandon liquid, every hole adds dimethyl sulfoxide 150 μ L; Jolt 10min under the room temperature; Blue crystallization is dissolved fully, and to detect wavelength 570nm, reference wavelength 655nm measures every hole OD value in Bio-Rad 450 type ELIASAs.
Do not add cell and do not add hyaluronic acid gold and be background, be 100% and suppress; Only add cell and do not add the negative contrast of hyaluronic acid gold, be 0% and suppress.
Hyaluronic acid gold suppression ratio %=(negative control OD-hyaluronic acid gold OD)/(negative control OD-background OD) * 100%.
As shown in table 4, suppression ratio wherein and standard deviation are percentile average of the inhibition of each parallel hole in every group of dosage and standard deviation.Result's matter acid gold that shows transparency has the propagation of A549 human lung carcinoma cell and suppresses active, and especially the propagation to the A549 human lung carcinoma cell has the edge to suppress active under high dose.
Table 4 hyaluronic acid gold is to the influence of A549 human lung carcinoma cell propagation
Figure BSA00000399255800091
Embodiment 7
(1) the hyaluronic acid gold 1mg that embodiment 1 is prepared adds a little dimethyl sulfoxide 200 μ L earlier makes its dissolving, and adding phosphate buffer again, to make the pH value of solution be 7.2, obtains the mother solution of hyaluronic acid gold.With 1640 culture medium that contain 10% serum, the mother solution of said hyaluronic acid gold is diluted to the working solution concentration of hyaluronic acid gold;
(2) human cervical carcinoma Hela cell is processed cell suspending liquid with 1640 culture medium that contain 10% serum, add 96 well culture plates (100 μ L/ hole), place 37 ℃, 5%CO by finite concentration 2Cultivate 24h in the incubator under the condition.Add the working solution of hyaluronic acid gold, its final concentration is respectively: 0 μ g/mL, 5 μ g/mL, 50 μ g/mL (8 multiple holes of every concentration).Discard culture fluid behind the effect 72h, every hole adds the RPMI-1640 (containing 10% serum) that 100 μ L contain 0.5mg/mL MTT, places 37 ℃, 5%CO 2Produce blue crystallization after cultivating 4h in the incubator under the condition, abandon liquid, every hole adds dimethyl sulfoxide 150 μ L; Jolt 10min under the room temperature; Blue crystallization is dissolved fully, and to detect wavelength 570nm, reference wavelength 655nm measures every hole OD value in Bio-Rad 450 type ELIASAs.
With the OD value that do not add hyaluronic acid gold 100% survival rate as human cervical carcinoma Hela cell, the survival rate %=of human cervical carcinoma Hela cell (OD/ that adds behind the hyaluronic acid gold does not add the golden OD of hyaluronic acid) * 100%.
Show that like table 5 result the hyaluronic acid gold has certain inhibition active to the propagation of human cervical carcinoma Hela cell.
Table 5 hyaluronic acid gold is to the influence of human cervical carcinoma Hela cell proliferation
Figure BSA00000399255800092
Embodiment 8
(1) the hyaluronic acid gold 1mg that embodiment 2 is prepared adds a little dimethyl sulfoxide 200 μ L earlier makes its dissolving, and adding phosphate buffer again, to make the pH value of solution be 7.4, obtains the mother solution of hyaluronic acid gold.With 1640 culture medium that contain 10% serum, the mother solution of said hyaluronic acid gold is diluted to the working solution concentration of hyaluronic acid gold;
(2) human cervical carcinoma Hela cell is processed cell suspending liquid with 1640 culture medium that contain 10% serum, add 96 well culture plates (100 μ L/ hole), place 37 ℃, 5%CO by finite concentration 2Cultivate 24h in the incubator under the condition.Add the working solution of hyaluronic acid gold, its final concentration is respectively: 0 μ g/mL, 5 μ g/mL, 50 μ g/mL (8 multiple holes of every concentration).Discard culture fluid behind the effect 72h, every hole adds the RPMI-1640 (containing 10% serum) that 100 μ L contain 0.5mg/mL MTT, places 37 ℃, 5%CO 2Produce blue crystallization after cultivating 4h in the incubator under the condition, abandon liquid, every hole adds dimethyl sulfoxide 150 μ L; Jolt 10min under the room temperature; Blue crystallization is dissolved fully, and to detect wavelength 570nm, reference wavelength 655nm measures every hole OD value in Bio-Rad 450 type ELIASAs.
With the OD value that do not add hyaluronic acid gold 100% survival rate as human cervical carcinoma Hela cell, the survival rate %=of human cervical carcinoma Hela cell (OD/ that adds behind the hyaluronic acid gold does not add the golden OD of hyaluronic acid) * 100%.
Show that like table 6 result the hyaluronic acid gold has certain inhibition active to the propagation of human cervical carcinoma Hela cell.
Table 6 hyaluronic acid gold is to the influence of human cervical carcinoma Hela cell proliferation
Figure BSA00000399255800101
The present invention includes but be not limited to above embodiment, every any replacement or local improvement of being equal to of under spirit of the present invention and principle, carrying out all will be regarded as within protection scope of the present invention.

Claims (4)

1. the purposes of a hyaluronic acid gold is characterized in that: the application of hyaluronic acid gold conduct preparation medicine for treating tumor thing.
2. the purposes of a kind of hyaluronic acid gold according to claim 1 is characterized in that: the hyaluronic acid gold is as the application of preparation treatment cancer drug.
3. the purposes of a kind of hyaluronic acid gold according to claim 1 is characterized in that: the hyaluronic acid gold is as the application of preparation treatment liver-cancer medicine.
4. the method for preparing of hyaluronic acid gold, it is characterized in that: concrete method for preparing step is following:
(1) Sodium Hyaluronate or hyaluronic acid are placed deionized water; Stirring at room solution makes it to dissolve fully and is transparence; Obtain Sodium Hyaluronate or hyaluronic acid solution; After the halogenation gold is dissolved in deionized water, obtain the halogenation gold solution, said halogenation gold solution is joined in said Sodium Hyaluronate or the hyaluronic acid solution; Stir; Room temperature reaction 8～24h stops to stir, and obtains reaction solution; Or the halogenation gold is dissolved in the deionized water, under stirring at room, add Sodium Hyaluronate or hyaluronic acid, till room temperature continues to be stirred to dissolving fully, stop to stir then, obtain reaction solution;
(2) said reaction solution is poured in the dehydrated alcohol below 0 ℃, produced thread deposition;
(3) said thread deposition is washed 3～4 times with decantation with the dehydrated alcohol below 0 ℃ or with using absolute ethanol washing behind the said thread sedimentation and filtration, the thread deposition after obtaining washing;
(4) the thread sedimentation and filtration after said the washing is gone out and below 40 ℃, after drying under the 0.09MPa vacuum condition, promptly obtain hyaluronic acid gold of the present invention;
Wherein, the halogenation gold is 1: 1 with the ratio of hyaluronate sodium or hyaluronic amount of substance in the step (1), and said halogenation gold refers to AuX 3, X be Cl, Br, I or F one of them;
The halogenation gold is following with the reaction equation of hyaluronate sodium:
3NaHA+3AuX 3→Au(HA) 3+3NaX+2AuX 3；
The dehydrated alcohol purity of using in the said method for preparing as AG or more than.
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Publication number Priority date Publication date Assignee Title
RU2534789C1 (en) * 2013-06-19 2014-12-10 Сергей Алексеевич Успенский Solid-phase method of production of water-soluble bioactive nanocomposite based on hyaluronic acid modified by citric acid and gold nanoparticles
CN116655825A (en) * 2023-06-02 2023-08-29 上海宜侬生物科技有限公司 Copper hyaluronate and preparation method and application thereof

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RU2684731C1 (en) * 2017-10-16 2019-04-12 Федеральное государственное бюджетное образовательное учреждение высшего образования "Кировский государственный медицинский университет" Министерства здравоохранения Российской Федерации (ФГБОУ ВО Кировский ГМУ Минздрава России) Method for preparing agent for local treatment of skin lesions based on gold nano-sized particles, ointment base and solid additives

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CN1760214A (en) * 2005-11-04 2006-04-19 山东福瑞达生物化工有限公司 Method for preparing transparent zinc hyaluronic acid

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CN1760214A (en) * 2005-11-04 2006-04-19 山东福瑞达生物化工有限公司 Method for preparing transparent zinc hyaluronic acid

Cited By (3)

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Publication number Priority date Publication date Assignee Title
RU2534789C1 (en) * 2013-06-19 2014-12-10 Сергей Алексеевич Успенский Solid-phase method of production of water-soluble bioactive nanocomposite based on hyaluronic acid modified by citric acid and gold nanoparticles
CN116655825A (en) * 2023-06-02 2023-08-29 上海宜侬生物科技有限公司 Copper hyaluronate and preparation method and application thereof
CN116655825B (en) * 2023-06-02 2024-06-18 上海宜侬生物科技有限公司 Copper hyaluronate and preparation method and application thereof

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